﻿﻿﻿Three novel species and new records of Kirschsteiniothelia (Kirschsteiniotheliales) from northern Thailand

﻿Abstract Kirschsteiniothelia (Kirschsteiniotheliales, Pleosporomycetidae) includes 39 saprobic species recorded from dead or decaying wood in terrestrial and freshwater habitats. This study focuses on exploring Kirschsteiniothelia diversity in woody litter in Thailand. Wood samples were collected from forest areas in Chiang Rai and Chiang Mai Provinces in Thailand and examined for fungal fructifications. Fungal isolates were obtained and their morphological and sequence data were characterised. Micromorphology associated with multilocus phylogeny of ITS, LSU and SSU sequence data identified three isolates as novel species (Kirschsteiniotheliainthanonensis, K.saprophytica and K.zizyphifolii) besides new host records for K.tectonae and K.xishuangbannaensis. The placement of the new taxa and records are supported by morphological illustrations, descriptions and molecular phylogenies and the implications of these findings are discussed. Our findings provide information for understanding Kirschsteiniothelia diversity and ecology.


Introduction
Since its introduction by Hawksworth (1985), the taxonomic placement of Kirschsteiniothelia (Kirschsteiniotheliaceae, Pleosporales, Pleosporomycetidae) has undergone several revisions.It was introduced in Pleosporaceae, with Kirschsteiniothelia aethiops as the type species.However, Barr (1993) moved it to Pleomassariaceae based on morphology and, based on molecular phylogenetic analyses, Schoch et al. (2006) demonstrated that K. aethiops does not belong to Pleosporaceae and should be placed in a new family.Kirschsteiniotheliaceae was established by Boonmee et al. (2012) to accommodate the holomorphic genus Kirschsteiniothelia.This was due to the fact that K. elaterascus and K. maritima clustered into Morosphaeriaceae and Mytilinidiales, respectively (Schoch et al. 2009;Suetrong et al. 2009;Boonmee et al. 2012).Later, Hernández-Restrepo et al. (2017) assigned it

Sample collection, fungal isolation and microscopic characterisation
Wood litter samples were collected from forest areas in Chiang Rai and Chiang Mai, Thailand.Morphological studies were performed following the methods described by Senanayake et al. (2020).The fungal structures were examined using a Leica EZ4 stereomicroscope.The micro-morphological features were observed and photographed using a Nikon ECLIPSE Ni compound microscope with a Canon 600 D digital camera.The Tarosoft Image Frame Work programme was used to measure specimen structures, and photo plates were prepared using the open-source Inkscape v.1.3(https://inkscape.org/).Pure cultures were obtained through single spore isolation on Difco potato dextrose agar (PDA) using the spore suspension method (Choi et al. 1999).Germinating spores were transferred to a new PDA plate and incubated at room temperature for seven days.Ex-type pure living cultures were deposited in the Mae Fah Luang University Culture Collection (MFLUCC) and herbarium material was deposited in the Mae Fah Luang University Fungarium (MFLU), Chiang Rai, Thailand.Faces of fungi numbers (FoF) (Jayasiri et al. 2015) and Index Fungorum numbers (Index Fungorum 2023) were obtained as instructed and the data were uploaded to the Greater Mekong Subregion in the GMS database (Chaiwan et al. 2021).

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from fresh mycelium scrapings using the EE.Z.N.A. Tissue DNA Kit from Omega Bio-tek, Inc., following the manufacturer's instructions.PCR amplifications were performed in a 50 μl reaction volume containing 10× PCR Master Mix, forward and reverse primers, DNA template and double sterilised H 2 O. Amplified DNA of the ITS, LSU and SSU were obtained through a polymerase chain reaction (PCR) using the pairs of primers ITS4/ITS5 (White et al. 1990), LROR/LR5 (Vilgalys and Hester 1990) and NS1/NS4 (White et al. 1990), correspondingly.The quality of the PCR products was visualised on a 1% agarose gel and sequenced by Biogenomed Co., Ltd (South Korea).

Alignments and phylogenetic analyses
The reads were assembled using the Staden Package (Staden et al. 2003) and compared against the NCBI non-redundant GenBank database (Sayers et al. 2020) and related reference sequences downloaded (Table 1).Except for concatenation and visualisation, all the steps of phylogenetic analysis were conducted in a Windows Subsystem for Linux (Microsoft, USA).The individual datasets were aligned using MAFFT with the --auto flag and automatically trimmed using TrimAl v.1.3with the -gt (0.3) option (Capella-Gutierrez et al. 2009).The best-fit model was selected using ModelTest-NG v.0.1.7 with the --template mrbayes option for DNA 3 schemes matrices (Darriba et al. 2020).The alignments were concatenated using SequenceMatrix and subjected to Maximum Likelihood (ML) and Bayesian Inference (BI) analyses.
Maximum Likelihood (ML) trees were generated using RAxML-HPC2 on XSEDE (8.2.8) (Stamatakis 2014) in the CIPRES Science Gateway platform (Miller et al. 2010), using 1,000 bootstraps replications and applying a partitioned model of evolution calculated by ModelTest-NG.Bayesian Inference was performed using MrBayes (Ronquist et al. 2012), with four simultaneous Markov Chain Monte Carlo (MCMC) chains and four runs for 3,000,000 million generations, sampling trees every 300 th generation.The first 25% of trees were discarded as burn-in and posterior probabilities (PP) were calculated from the remaining trees.The consensus phylograms were visualised using FigTree (Rambaut 2012) and edited using the open-source Inkscape v.1.3(https:// inkscape.org/).The newly-generated sequences are indicated in bold."T" refers to holotype or ex-type strains and "-" shows unavailable data in GenBank.
Culture characteristics.Conidia germinated on PDA within 48 hours.Germ tubes germinated from end cell.Colony, reaching 30-35 mm diam.after one month at room temperature, circular form, flat, undulate edges, dense velvety surface, dark green on the surface, white mycelium on the tip, dark in reverse with dark green margin.

Kirschsteiniothelia tectonae
Culture characteristics.Conidia germinating on PDA within 24 hours, reaching up to 15-20 mm diam.after one week at room temperature.Germ tubes generated from basal cells.Colony on PDA, dense, circular, flat or effuse, velvety, from front brown at the centre and black at the edge, from reverse, dark brown.Known distribution.Thailand (Li et al. 2016; this study) Known hosts.Tectona grandis (Li et al. 2016), Microcos paniculata and Dipterocarpus alatus (this study) Culture characteristics.Conidia germinating on PDA within 24 hours reaching up to 2 cm diam.after one week at room temperature.Germ tubes generated from both end cells.Colony on PDA, dense, circular, flat or effuse, velvety, from front, brown at the centre and dark brown at edge, from reverse, black to pale brown radiating.

Discussion
This study introduces three new species and new host records of Kirschsteiniothelia from dead wood from Chiang Rai Province, Thailand, based on morphological and molecular analyses (Figs 1-7).Kirschsteiniothelia species have been found almost worldwide, including in the United States of America (Hawksworth 1985;Hyde 1997;Wang et al. 2004;Su et al. 2016), Iran (Mehrabi et al. 2017), Switzerland (Hawksworth 1985;Wang et al. 2004), Thailand (Boonmee et al. 2012;Li et al. 2016;Bao et al. 2018;Hyde et al. 2018;Sun et al. 2021;Jayawardena et al. 2022), South Africa (Marincowitz et al. 2008), China (Chen et al. 2006;Su et al. 2016;Bao et al. 2018;Liu et al. 2023;Yang et al. 2023;Xu et al. 2023), Canada (Hawksworth 1985), Italy (Wang et al. 2004), Spain (Rodríguez-Andrade et al. 2019) and India (Bao et al. 2018).Most of the species (K.acutispora, K. chiangmaiensis, K. crustacea, K. emarceis, K. extensa, K. lignicola, K. phoenicis, K. rostrata, K. septemseptata, K. spatiosa, K. tectonae and K. thailandica) have been reported from Thailand (Boonmee et al. 2012;Li et al. 2016;Bao et al. 2018;Hyde et al. 2018;Sun et al. 2021;Jayawardena et al. 2022), representing more than 25% of the species in this genus.Our results expand the knowledge of the diversity of this genus, especially in Thailand.This genus is also prone to be highly speciose, given the recent introduction of ten new species (Jayawardena et al. 2022;Hyde et al. 2023;Liu et al. 2023;Louangphan et al. 2023 (under review); Xu et al. 2023).With the introductions of the present study (K.inthanonensis, K. saprophytica, K. paniculata and K. zizyphifolii), 32.5% of the species will have been introduced within two years, mainly as saprobes in woody litter.Besides, most Kirschsteiniothelia species have been reported from terrestrial environments, with only a few (K.cangshanensis, K. fluminicola and K. rostrata) reported from freshwater habitats (Bao et al. 2018).Their ecological significance also relies on their ability to infect humans (Nishi et al. 2018;Guegan et al. 2021).This demonstrates the potential for further discoveries on the diversity and lifestyles within Kirschsteiniothelia. Thus, exploring its diversity, especially in woody litter in protected environments and other tropical areas, will reveal the vast diversity within Kirschsteiniotheliaceae.For example, frequent incursions into fungal diversity have established Thailand as a hotspot for its diversity (Hyde et al. 2018).
Furthermore, Kirschsteiniothelia species appear to not have host specificity, as from our results, the same species were found associated with different hosts: K. xishuangbannaensis, previously reported from dead branches of Hevea brasiliensis (Xu et al. 2023), was recorded from Microcos paniculata (MFLUCC 23-0273) and Dipterocarpus alatus (MFLUCC 23-0274); K. paniculata was isolated from Microcos paniculata (MFLUCC 23-0271) and Dalbergia cana .In this regard, the host-specificity or host-recurrence of saprobic fungi has been discussed over the last two decades (Hooper et al. 2000;Zhou and Hyde 2001;Santana et al. 2005;Kodsueb et al. 2008;Tennakoon et al. 2022).However, saprotrophs seem to be less host-specific when compared with other trophic modes (Zhou and Hyde 2001).This may be because different hosts have different chemical compositions, which may affect the fungi of a particular species (Hyde et al. 2007).This hypothesis suggests that woody litter may harbour many species yet to be discovered (Kodsueb et al. 2008).
A combined approach should be employed to resolve the taxonomic placement of new species in this genus.This approach should include at least molecular phylogeny and morphological characters (Chethana et al. 2021;Maharachchikumbura et al. 2021).It should also include the linking of sexual and asexual morphologies, which are important factors in the taxonomy of Ascomycota, as pleomorphism can bias the morphological characters (Maharachchikumbura et al. 2021).However, only a few of the 39 Kirschsteiniothelia species, specifically K. atra and K. recessa (Hawksworth 1985) and K. lignicola and K. emarceis (Boonmee et al. 2012), are known from both their sexual and asexual morphs.
The findings of this study underscore the importance of integrating multiple types of evidence for the identification and classification of fungal species and they demonstrate the potential for further discoveries within Kirschsteiniothelia.The discovery of new species and host records has significant implications for our understanding of the ecological roles and interactions of this genus.In particular, identifying new host records provides valuable insights into the host range and specificity of Kirschsteiniothelia species, which may help elucidate the mechanisms underlying these interactions.Further research is necessary to fully explore the ecological significance of these findings and determine the potential impacts of Kirschsteiniothelia species on their hosts and ecosystems.

Figure 1 .
Figure 1.Maximum Likelihood phylogenetic tree generated from ITS, LSU and SSU sequence data for selected Kirschsteiniotheliales and related Dothideomycetes orders.The tree is rooted with Stemphylium vesicarium (CBS 191.86 and MFLUCC 14-0920).Newly-generated sequences are in blue and new species are in bold.Holotype and ex-type strains are symbolic by " T ".Maximum Likelihood bootstrap (MLBS) values ≥ 70% and Bayesian posterior probabilities (BYPP) ≥ 0.95 are shown at the nodes.

Table 1 .
Names, strain numbers, and corresponding GenBank accession numbers of Kirschsteiniotheliales taxa used in the phylogenetic analyses.

Table 2 .
Maximum Likelihood indices of Kirschsteiniothelia tree.Figs 2-7), three new species (K.zizyphifolii, K. inthanonensis and K. saprophytica) are proposed, along with the two new host records for K. xishuangbannaensis and K. tectonae.